The hematopoietic stem and progenitor cells (HSPCs) are the progenitor cells for all blood cells. The proliferation and differentiation of HSPCs give rise to the entire hematopoietic system. HSPCs are believed to be capable of self-renewal expanding their own population of stem cells and being pluripotent are capable of differentiating into any cell in the hematopoietic system. From this rare cell population, the entire mature hematopoietic system, comprising lymphocytes (B and T cells of the immune system) and myeloid cells (erythrocytes, megakaryocytes, granulocytes and macrophages) are formed. The lymphoid lineage, comprising B cells and T cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. The myeloid lineage, which includes monocytes, granulocytes, megakaryocytes as well as other cells, monitors for the presence of foreign bodies, provides protection against neoplastic cells, scavenges foreign materials, produces platelets, and the like. The erythroid lineage provides red blood cells, which act as oxygen carriers.
Various conditions in which HSPCs are depleted in the human body include leukemias, lymphomas, myelomas, different haematological disease such as phagocyte disorders, anaemia, viral diseases and external factors such as exposure to ionizing radiation. Hematopoietic stem and progenitor cells (HSPCs) are very sensitive to ionizing radiation and show massive apoptosis (˜50%) at 1 Gy dose of γ-radiation. Exposure to higher doses of radiation induces severe depletion of HSPC and mitotic arrest. In order to reduce radiation toxicity to normal tissues during planned exposure, radiotherapy is delivered to tumours while minimizing the volume of normal tissue using different physical techniques. However, considering the anatomical location and unclear boundary between tumour and the surrounding normal tissue, normal tissue toxicity remains a major concern.
Several studies have been performed with objective to develop new and effective countermeasures to reduce, prevent or lower the risk of normal tissue radiation toxicity. Several drug targets such as free radicals, prosurvival transcription factors NF-κB and Nrf-2, cytokine receptors, toll-like receptors, and radical scavengers have been proposed. (Gudkov A. V. et al., J ClinInves, 120:2270-2273, 2010; Khan N. M. et al., Free RadicBiol Med, 51:115-128, 2011; and Tekmura N. et al., Nat Commun, 5:3492, 2014).
Amifostine was identified as a free radical scavenger and remains as the best drug tested as a radio-protector in clinic. It has been shown to prevent radiotoxicity in mucosal tissue in patients undergoing external radiotherapy for head and neck cancer, pelvis or brachytherapy for cervical cancers, and for patients undergoing radioiodine therapy (Kouvaris J. R. et al., Oncologist, 12:738-747, 2007). Several other molecules such as naphthoquinone, γ-tocotrienol, genestein, 3, 3′-diindolylmethane, and simvastatin which target cellular signaling machinery have been shown to exhibit potential radio-protective effects in experimental systems (Barbee M. et al., Radiat Res, 171:596-605, 2009 and Landauer M. R. et al., J ApplToxicol, 23:379-385, 2003). A flagellin-derived polypeptide CBLB502 which binds to toll-like receptor 5 (TLRS) and activates NF-κB in enterocytes and intestinal endothelial cells was discovered as both a radioprotector and a mitigator of radiation injury in mouse models (Burdelya L. G. et al., Science, 320:226-230, 2008). However, there is controversy over the radioprotective effects of these drugs towards tumour cells also. Moreover, until now, no radioprotective agent is clinically approved for prevention of radiation induced hematopoietic syndrome.
Cancer stem cells (CSCs) are resistant to chemotherapy and radiotherapy and are responsible for relapse of cancer in more than 50% of the patients. Recent observations suggest that it will be imperative to target all CSC subsets within the tumour to prevent relapse (Visveder and Lindeman. Cell Stem Cell, 10:717-728, 2012).
In spite of various methods known in the art to increase in vivo abundance of HSPCs, there still remains a need for new methods or compositions which can be used in conditions involving depletion of HSPCs. The present invention provides a pharmacological composition for specifically targeting and killing the cancer cells and particularly cancer stem cells while protecting the normal stem cells (HSPCs) during radiotherapy. The FDA-approved drugs, amifostine and palifermin are used for preventing the side effects of radiotherapy in cancer patients. However, these drugs per se have severe side effects and their protective action is not limited to the normal tissues.
Hence, there is a need to develop agents, which would specifically protect normal tissues, but not tumours and/or sensitize the tumour cells to radiation.
Chlorophyllin is a constituent of the over the counter drug Derifil and it falls under the class of GRAS (grossly recognized as safe) drugs. It has been earlier shown to scavenge free radicals derived from radiation. Chlorophyllin was also shown to act as an antioxidant and thereby prevent radiation-induced damage to biomolecules like DNA in cell free systems. Chlorophyllin was also shown to enhance the immune responses in mice. There are also reports that chlorophyllin inhibited radiation induced micronuclei induction and sister chromatid exchanges. However there are also reports showing that it did not prevent radiation induced DNA damage in mice (Mutat Res. 1996 February, (367(2):51-6).
It is not obvious that an antioxidant should protect against radiation induced morbidity and mortality. Several antioxidants shown to reduce radiation induced micronuclei but there are no reports on these antioxidants protecting against radiation-induced mortality eg. Gallic acid, caffeic acid, vitamin C, vitamin E, glutathione, N-acetyl cysteine are all well-known antioxidants reported to reduce radiation induced micronuclei formation. However, only few of them offer protection against radiation induced mortality. Bilirubin is one of the most effective antioxidants but reported to enhance radiation induced mortality in mice (Free Radic Biol Med. 2012 Sep. 1; 53(5): 1152-69).
Several pro-oxidants like 1,4-Naphthoquinone have been shown to act as effective radioprotectors (Free Radic Biol Med. 2011 Jul. 1; 51(1):115-28). Granulocyte-Colony stimulation factor is an FDA approved radioprotector and it induces reactive oxygen species.
Effects of chlorophyllin on stem cells other than HSPC have been studied by Yin Li-ming et al. The study revealed the effect of sodium copper chlorophyllin on the proliferation, differentiation and immunomodulatory function of mesenchymal stem cells (MSCs) from mice with aplastic anaemia (Yin Li-ming et al., Chin J Integr. Med, 19(5):360-366, 2013). Yin Li-ming et al have shown the effect of a dose range of 25, 50 or 100 mg/kg body weight given to mice for 20 consecutive days after exposure of mice to 5 Gy dose of gamma irradiation. In their publication, there is no data on survivability of mice and the dose of radiation used is below the lethal dose. Increased MSC count cannot be correlated with increased survival after exposure to lethal dose of radiation. Further, the treatment regimen proposed by Yin-Li-Ming et al shows that the MSC can suppress immune responses, however, the regimen proposed by us shows more robust immune response (increased neutrophil counts) and faster recovery from radiation induced bone marrow aplasia. Yin et al have not investigated the effect of chlorophyllin on radiation induced hematopoietic syndrome. Thus, there are no reports until now on the radioprotecting effect of chlorophyllin on HSPCs and its therapeutic methodologies in relation to hematopoietic stem and progenitor cells (HSPCs) and radiotherapy for better therapeutic outcome.